特殊植化物對於人類單核球細胞株(THP-1)在LPS刺激下之功能性基因組研究

Abstract

在人類免疫系統中，單核球 (monocyte) 參與體內第一線的防禦機制，並被認為是具有可分化為樹突狀細胞 (dendrite cell) 或巨噬細胞 (macrophages) 能力的前驅細胞。單核球平時在血液系統中移動，一旦受到外來的刺激，一方面會分泌許多調節免疫功能的細胞激素（cytokines），同時亦會移動到目標組織中，進而分化為巨噬細胞。在先天免疫反應中單核球或巨噬細胞都扮演重要的角色，它們所分泌出來的細胞激素如： TNF-α、IL-1、IL-6 ，可以造成急性期反應 (acute-phase response)，並啟動後續與發炎反應有關的訊息傳導路徑。目前在我們實驗室中已經有許多植物化合物具有抗發炎的活性；例如在紫草中的紫草素 (shikonin) 被證實可以抑制 TNF-α mRNA 的表現；紫錐菊粗萃物中的正丁醇層（Butanol fraction）似乎在樹突狀細胞之微矩陣實驗中具有抗發炎的活性；大黃中的大黃素 (emodin) 則可以降低樹突細胞中有關於發炎反應的表面抗原的生成；咸豐草中的萃取物(cytopiloyne) 在單核球細胞株中，似乎具有降低TNF-α及IL-6 mRNA表現的活性。目前我們對於這些植化物如何調節影響免疫反應的機制並不清楚，因此我們意圖經由利用單核球細胞株（THP-1）在脂多醣體（LPS）刺激下，利用由228個與免疫相關的基因組成DNA晶片，在不同的時間點中去測定這些基因mRNA的表現。期能以系統性的方式來檢視免疫反應的機轉。在本次實驗中我們發現紫草素在0.5小時的時候可以降低在LPS刺激下會表現的某些與發炎反應有關基因之mRNA的表現，例如：TNF-α、IL-1β、IL-8、CCL4、CCL5以及PTGS1、2。而大黃素則在0.5、2、24、48小時也具有抑制一些與發炎有關的基因的表現。在cytopiloyne的處理下， TNF-α、IL-8 、CCL3、CCL4、CCL5、CD14、STAT1、IL-1β在後期的表現量增加外，其它的時間點都觀察到與發炎反應有關的某些特殊基因的表現量降低。至於紫錐菊粗萃物中的正丁醇層，除了CCL3、STAT1、IL-1β、IFNAR1在八小時或48小時基因表現有增加外，其它的時間點都觀察到與發炎反應有關的基因的表現量降低。此外我們也發現紫草素及大黃素對於NFATC基因具有很強的抑制作用，而NFATC是IL-2的轉錄因子，IL-2可以刺激B細胞與T細胞的增生，因此紫草素及大黃素可能在活體中具有調節B細胞與T細胞增生的功能。Cytopiloyne對於WNT1基因具有很強的抑制作用，而前人曾研究指出WNT1過度表現時會造成染色體數目異常，是一種oncogene。我們希望在未來能夠作更進一步的分析，去確定這些植化物在動物體內對單核球是否具有相同的活性，期望能藉此更進一步評估這些植化物是否具有醫療的效果。

In human systems, monocytes can differentiate into dendritic cells (DCs) or macrophages, and respond dramatically to a number of immune-modulation activities. Both monocytes and macrophages are known as important cell types that play a key role in innate immunity and produce a number of cytokines, including tumor necrosis factor alpha (TNF-α), interleukine-1β (IL-1β) and interleukine-6 (IL-6). These cytokines can induce “systemic acute-phase immune responses” which are often involved in pro-inflammatory signaling pathways. In our laboratory, we have accumulated a series of phytocompounds that confer anti-inflammatory activities. For example, shikonin can decrease TNF-α mRNA expression. Cytopiloyne can decrease monocytes’ TNF-α and IL-6 mRNA expression in RT-PCR assay. Emodin can substantially decrease specific DC surface marker that is associated with inflammation. The Echinacea purpura butanol fraction seems to confer in anti-inflammatory response in DCs. Based on the above observations, we evaluated the immune responses in terms of differential gene expressions in LPS-stimulated THP-1 cells, a monocyte cell line, by use of home-made mini DNA microarray chips which contains 228 genes. In this study, shikonin can decrease expression of genes encoding some LPS-induced signaling pathways, including TNF-α, IL-1β, IL-8, cysteine-cysteine motif chemokine ligand (CCL4), CCL5 and prostaglandin synthase (PTGS) gene at 0.5 hr. Emodin also can decrease TNF-α, IL-1β, IL-8, CCL4, CCL5 and PTGS gene expression at 0.5, 2, 24 and 48 hrs post treatment. Cytopiloyne can decrease expression of genes encoding LPS-induced signaling pathways at all time points tested, TNF-α, IL-8, CCL3, CCL4, CCL5, CD14, STAT1 and IL-1β were increased in mRNA expression at 24 and 48 hrs. Echinacea can decrease the expression of genes that encode LPS-induced signaling pathways at all time points tested, CCL3, STAT1, IL-1β and IFNAR1 were increased in mRNA expression at 8 and 48 hrs. Besides, we found shikonin and emodin conferred strong effect on NFATC gene expression. NFATC gene is a transcriptional factor of IL-2, a cytokine that can stimulate T and B cells proliferation. Therefore, shikonin and emodin may be able to effect T and B cell proliferation. Cytopiloyne had a strong effect on oncogene, WNT1 gene, via down-regulation. In future studies, we intend to use applicable in vivo system to study the effect of these phytocompounds using animal models, and hope to employ these phytocompounds as useful food supplements or alternative herbal medicines.